The soluble guanylyl cyclase (sGC) is a key signaling protein that stimulates the production of the second messenger cGMP when activated by nitric oxide. This cGMP signaling pathway is important for a number of cardiovascular processes including blood pressure regulation and sGC has therefore attracted considerable attention as a pharmaceutical drug target to treat cardiovascular diseases such as hypertension, heart failure, atherosclerosis, erectile dysfunction, thrombosis, and renal fibrosis. Several compounds targeting sGC are in pre-clinical or clinical trial stages. The applicant has aided the structural understanding of guanylyl cyclases by determining crystal structures of 4 different domains including complexes with the sGC activators BAY 58-2667 and nitric oxide. The overarching goal of this application is to further unravel the signaling intricacies of sGC that could yield insights into how to either utilize or bypass this signaling mechanism such that this information can be pharmaceutically exploited to modulate sGC. The proposed research is of a highly collaborative and multi-disciplinary nature involving techniques such as X-ray crystallography, electron paramagnetic resonance (EPR), isothermal calorimetry (ITC), mutagenesis, and cell biology/activity measurements. The 3 Specific Aims are: Specific Aim 1: To test the hypothesis that the activation of the H-NOX domain of sGC involves a partial distortion of the heme pocket. The activation by sGC activators will also be probed for comparison and to delineate the mode of action of these heme mimetics. Specific Aim 2: To test the hypothesis that the coiled-coiled (CC) domain of sGC is a parallel dimer and that this domain is not static and undergoes conformational changes during the activation process. Specific Aim 3: To test the hypothesis that the N-terminal half of sGC is responsible for binding sets of chemically distinct sGC stimulators; this sGC region includes the H-NOX and PAS/H-NOXA domain(s). The proposed research will lead to new molecular insights into how sGC functions and how GC activators/modulators work and this will enhance our understanding of cardiovascular processes and could lead to the development of new drugs to treat cardiovascular diseases.